Palmitoylation and depalmitoylation of cysteine residues on target proteins plays a key role in regulating protein localization, trafficking, and resultant cell signaling. See Linder and Deschenes (2007) Nat Rev Mol Cell Biol 8, 74-84; Eisenberg et al. (2013) Biochem Soc Trans 41, 79-83; Topinka and Bredt (1998) Neuron 20, 125-134; and Chan et al. (2016) Nat Chem Biol 12, 282-289. Recent advances in mass spectrometry-based protein profiling techniques have greatly expanded the catalog of human proteins modified by S-palmitoylation to include hundreds of putative targets. See Peng et al. (2016) Curr Opin Chem Biol 30, 77-86; and Hernandez et al. (2013) Curr Opin Chem Biol 17, 20-26. There are 23 protein acyltransferases (PATs) in humans that act as the “writers” that install the lipid modification. See Zhenq et al. (2013) J Am Chem Soc 135, 70782-7085. Acyl protein thioesterases (APTs) are the “erasers” of S-palmitoylation (see Linder and Deschenes (2007) Nat Rev Mol Cell Biol 8, 74-84), including the lyosomal degradation enzyme protein palmitoylthioesterase-1 (see Verkruyse and Hofmann (1996) J Biol Chem 271, 15831-15836) and the putatively cytosolic proteins acyl protein thioesterase 1 (APT1) and acyl protein thioesterase 2 (APT2), which are part of the metabolic serine hydrolase (mSH) superfamily. See Long and Cravatt (2011) Chem Rev 111, 6022-6063. In addition, three previously uncharacterized enzymes, the α/β-hydrolase domain-containing protein 17 members A-C, were recently identified as regulators of N-Ras depalmitoylation and are potentially additional S-palmitoylation erasers. See Lin and Conibear (2015) Elife 4, e11306.
APT enzymes have been proposed to contain no substrate specificity and to act constitutively to depalmitoylate proteins universally from intracellular membranes. See Rocks et al. (2010) Cell 141, 458-471. However, several lines of evidence suggests that S-palmitoylation is, in fact, regulated and dynamic, including findings that PSD-95 is rapidly depalmitoylated following glutamate stimulation (see EI-Husseini et al. (2002) Cell 108, 849-863), that activation of FGF receptors by FGF2 leads to palmitoylation of cell adhesion molecules in neurons (see Ponimaskin et al. (2008) J Neurosci 28, 8897-8907), and that T-cell activation induces accelerated palmitate cycling. See Zhang et al. (2010) Proc Natl Acad Sci USA 107, 8627-8632. Indeed, the APT enzymes themselves can be S-palmitoylated (see Kong et al. (2013) J Biol Chem 288, 9112-9125), potentially resulting in auto-regulatory mechanisms. To address the roles of APT activities in cell signaling, several classes of small molecule inhibitors have been developed, including the pan-depalmitoylase inhibitor palmostatin B (PalmB) and the APT1- and APT2-specific inhibitors ML348 and ML349. See Davda and Martin (2014) Medchemcomm 5, 268-276; Dekker et al. (2010) Nat Chem Biol 6, 449-456; Rusch et al. (2011) Angew Chem Int Ed Engl 50, 9838-9842; and Adibekian et al. (2012) J Am Chem Soc 134, 10345-10348.
Current approaches to study S-palmitoylation primarily involve proteomics, monitoring the trafficking of microinjected fluorescent substrates (see Dekker et al. (2010) Nat Chem Biol 6, 449-456; and Gormer et al. (2012) Chembiochem 13, 1017-1023), or the use of cell-permeable substrate mimetics. See Creaser and Peterson (2002) J Am Chem Soc 124, 2444-2445. These approaches reveal the balance between palmitoylation and depalmitoylation, but cannot reveal potential upstream regulatory mechanisms particularly related to the depalmitoylases.
Accordingly, there is an ongoing need for further methods for studying S-palmitoylation and other PTMs of cysteines. More particularly, there is an ongoing need for additional methods to study the regulation and dynamics of S-palmitoylation, such as methods that can monitor the activity levels of depalmitoylases in physiological relevant contexts, such as in live cells and heterogeneous tissues. There is also a need for additional synthetic molecular probes that can detect the activity of depalmitoylases and related enzymes. For example, there is the need for synthetic probes containing chemical linkers that can be cleaved in the presence of depalmitoylases or ratiometric probes that can undergo a change in detectable properties in the presence of depalmitoylases. There is also the need for synthetic molecular probes with good water solubility.